Detection of Candida in concentrated oral rinse cultures by real-time PCR

The incidence of oral candidosis has increased in recent years, largely as a result of the emergence of human immunodeficiency virus infection and the more widespread use of immunosuppressive chemotherapy. This development has been associated with a need for more reliable methods for the detection of Candida. The present study assessed the performance of a real-time PCR and two block-based PCRs for the detection of Candida in 193 concentrated oral rinse culture (CRC) specimens. A total of 102 CRC specimens were positive by culture for Candida; and 96, 90, and 75 of these were also positive by real-time, N18-specific, and internal transcribed spacer (ITS)-specific PCRs, respectively. The five false-negative results by the real-time PCR were all non-Candida albicans positive by culture. Of the 91 culture-negative CRC specimens, 20, 41, and 44 were positive by the real-time PCR and the N18- and ITS-specific PCRs, respectively. All three PCRs detected fungal DNA in 8 culture-negative CRC specimens, with a further 30 being positive by two of the three PCRs. A total of 32 CRC specimens were Candida free by all methods. In summary, a real-time PCR that provides a sensitive, specific, and rapid alternative technique for detection of Candida in the mouth is described.     

The Effect of End Group Modification on the Transparency of Vinyl Addition Norbornene Polymers at 193 nm

Summary: Homopolymers of a bis‐trifluorocarbinol substituted norbornene ( 1 ) (α,α‐bis(trifluoromethyl)bicyclo[2.2.1]hept‐5‐ene‐2‐ethanol or HFANB) and copolymers of 1 with t‐butyl ester of 5‐carboxylic acid ( 2 , t‐BuEsNB) were produced using palladium catalysts and olefinic chain transfer agents such as 1‐hexene and ethylene to control molecular weight. However, these low‐molecular‐weight polymers exhibited relatively low optical transparencies at 193 nm. In fact, the opacity (measured as optical densities in absorbance units per micron) of thin films of these homo‐ and co‐polymers was inversely proportional to their molecular weight. This relationship is consistent with an end group contribution to the film opacity. Spectroscopic analysis of these polymers by ¹H NMR and MALDI‐TOF MS confirmed that 1‐hexene and ethylene chain transfer agents generated olefin‐terminated vinyl addition polymers. The olefinic end group contribution to optical density can be eliminated by appropriate chemical modification. Both epoxidation and hydrogenation of the polymer olefinic end groups generated very low optical density materials, independent of molecular weight, that are suitable as 193‐nm photoresist binder resins.

End group modification of vinyl and hexenyl‐terminated homopolymers of 1 by epoxidation or hydrogenation.     

Inhibition of interleukin-8 expression by dexamethasone in human cultured airway epithelial cells.

Interleukin-8 (IL-8) is a neutrophil chemotactic factor expressed in many cell types, including human airway epithelial cells (HAEC). Inhaled corticosteroids are now used increasingly early in the treatment of airway inflammation such as in asthma, and directly interact with HAEC at relatively high concentrations. We have investigated the effect of dexamethasone on IL-8 expression in primary cultured HAEC obtained from transplantation donors. Northern blot analysis was used to measure IL-8 mRNA levels in HAEC, and radioimmunoassay was used to measure IL-8 protein in culture supernatant fluids. We demonstrated that IL-8 was expressed by primary cultured HAEC and that this was enhanced by IL-1 beta and tumour necrosis factor-alpha stimulation, but not by IL-6 or lipopolysaccharide. Dexamethasone suppressed IL-8 mRNA expression and protein synthesis dose-dependently in both resting and stimulated HAEC. The half-life of IL-8 mRNA determined in the presence of actinomycin D was less than 1 hr, and dexamethasone preincubation had no effect on mRNA stability. These results support the view that HAEC may play an important role in the pathogenesis of airway inflammatory diseases, and that glucocorticosteroids may exert their anti-inflammatory effects by blocking IL-8 gene expression and generation in these cells.     

Distribution of an antigenically related iron-regulated protein among the Neisseria spp.

Several iron-regulated proteins of Neisseria gonorrhoeae have been reported. One of these, a 37,000-molecular-weight protein (37K protein), appears to be common to all gonococcal isolates. Recently, the occurrence of a similar protein has also been noted in N. meningitidis. The gonococcal 37K protein has been purified and used to produce both rabbit monospecific antiserum and murine monoclonal antibodies. Using these antibody reagents, we analyzed 57 strains from nine species of Neisseria and the closely related organism Branhamella catarrhalis for the presence of proteins antigenically related to the gonococcal 37K protein. Strains grown on medium with low iron content were probed for antigenic reactivity by Western blot techniques and an enzyme-linked immunosorbent assay. Proteins which cross-reacted with the rabbit monospecific antiserum were designated as AgR-37K proteins. The data indicated that the AgR-37K proteins were conserved among the 40 strains of N. gonorrhoeae, N. meningitidis, N. lactamica, and N. cinerea tested. Seventeen strains from other species of Neisseria and Branhamella did not express AgR-37K proteins with the exception of one N. subflava isolate. All AgR-37K proteins appeared to be regulated by the amount of available iron in the growth medium. Murine monoclonal antibodies were used to probe the antigenic heterogeneity of the AgR-37K proteins from different Neisseria spp. Two of seven monoclonal antibodies were broadly cross-reactive, recognizing the AgR-37K proteins from all species examined. The remaining five monoclonal antibodies were more discriminating, recognizing the AgR-37K proteins from certain species. The antigenic conservation of these AgR-37K proteins, particularly among the pathogenic members of the genus Neisseria, may imply that these proteins serve a common function in pathogenicity.     

Absence of a prominent Th2 cytokine response in human tuberculosis.

Depressed Th1 responses are a prominent feature of human tuberculosis, but an enhanced Th2 response has not been detected in peripheral blood T cells stimulated in vitro with Mycobacterium tuberculosis. In disease due to Mycobacterium leprae, Th2 cells predominate in tissue lesions of patients with extensive disease but are absent from peripheral blood. To determine if Th2 cells are present in tissue lesions of tuberculosis patients, we evaluated patterns of cytokine expression in lymph nodes from tuberculosis patients with or without human immunodeficiency virus infection and in controls without tuberculosis. Gamma interferon and interleukin-10 (IL-10) mRNA expression in tuberculosis patients with or without human immunodeficiency virus infection was high, whereas IL-4 expression in the same patients was low. Immunolabeling studies showed that macrophage production of IL-12 was increased in lymph nodes from tuberculosis patients, that gamma interferon was produced by T cells, and that IL-10 was produced by macrophages rather than Th2 cells. These results indicate that Th2 responses are not enhanced either systemically or at the site of disease in human tuberculosis.     

Hippocampal granule cells are necessary for normal spatial learning but not for spatially-selective pyramidal cell discharge

Summary The effects of massive destruction of granule cells of the fascia dentata on the spatial and temporal firing characteristics of pyramidal cells in the CA1 and CA3 subfields of the hippocampus were examined in freely moving rats. Microinjections of the neurotoxin colchicine were made at a number of levels along the septo-temporal axis of the dentate gyri of both hemispheres, resulting in destruction of over 75% of the granule cells. By contrast there was relatively little damage to the pyramidal cell fields. As assessed by three different behavioral tests, the colchicine treatment resulted in severe spatial learning deficits. Single units were recorded from the CA1 and CA3 subfields using the stereotrode recording method while the animals performed a forced choice behavioral task on the radial 8-arm maze. Considering the extent of damage to the dentate gyrus, which has hitherto been considered to be the main source of afferent information to the CA fields, there was remarkably little effect on the spatial selectivity of place cell discharge on the maze, as compared to recordings from control animals. There was, however, a change in the temporal firing characteristics of these cells, which was manifested primarily as an increase in the likelihood of burst discharge. The main conclusion derived from these findings is that most of the spatial information exhibited by hippocampal pyramidal cells is likely to be transmitted from the cortex by routes other than the traditional trisynaptic circuit. These routes may include the direct projections from entorhinal layers II and III to CA3 and CA1, respectively.     

Density heterogeneity of eosinophil leucocytes: induction of hypodense eosinophils by platelet-activating factor.

We have examined the induction of hypodense eosinophils by platelet-activating factor (PAF), a mediator which may be involved in eosinophil activation in allergic diseases. Guinea-pig eosinophils were incubated with buffer or PAF and applied to continuous Percoll density gradients. Cellular density ranged from 1.0142 to 1.1369 g/ml. Peak eosinophil density in control was 1.0887 +/- 0.0008 g/ml (mean +/- SEM), and 91.1 +/- 1.4% of eosinophils were distributed between 1.0810 and 1.1000 g/ml. Preincubation of eosinophils with PAF(10(-7) M) resulted in a time-dependent and non-cytolytic increase of the number of hypodense eosinophils, with peak densities after incubation for 1 hr and 2 hr of 1.0834 +/- 0.0014 (n = 4, P less than 0.05) and 1.0755 +/- 0.0007 g/ml (n = 6, P less than 0.01), respectively. After incubation for 2 hr, 82.0 +/- 4.9% (n = 6) eosinophils showed a density lower than 1.080 g/ml. Lyso-PAF, the inactive precursor and metabolite of PAF, at a concentration of 10(-7) M had no effect on cell density. The specific PAF receptor antagonist WEB 2086 (10(-6) M) inhibited the PAF-induced density shift by 87.0 +/- 5.3%. Our results demonstrate that a single mediator is able to induce the formation of hypodense eosinophils. We conclude that the appearance of hypodense eosinophils in allergic diseases such as asthma may occur, at least in part, in response to inflammatory mediators which activate these cells.     

Quantitative culture of endocervical Chlamydia trachomatis.

We examined the number of Chlamydia trachomatis inclusions produced in the initial passage of cell cultures of endocervical specimens from 1,231 women with positive chlamydial cultures who attended a sexually transmitted diseases clinic. Youth, white race, oral contraceptive use, and concurrent infection by Neisseria gonorrhoeae were associated with high chlamydial inclusion counts. Youth, white race, and oral contraceptive use were independent determinants of a high chlamydial inclusion count in women without concurrent gonorrhea but not in women with gonorrhea. Results of our study suggest that the degree of chlamydial excretion from the infected cervix may be influenced by characteristics of the patient being tested and may affect the ability to detect C. trachomatis in different patient groups.     

Laboratory diagnosis of human chlamydial infections.

Chlamydia trachomatis is a human pathogen that causes ocular disease (trachoma and inclusion conjunctivitis), genital disease (cervicitis, urethritis, salpingitis, and lymphogranuloma venereum), and respiratory disease (infant pneumonitis). Respiratory chlamydioses also occur with infection by avian strains of C. psittaci or infection by the newly described TWAR agent. Diagnosis of most acute C. trachomatis infections relies on detection of the infecting agent by cell culture, fluorescent antibody, immunoassay, cytopathologic, or nucleic acid hybridization methods. Individual non-culture tests for C. trachomatis are less sensitive and specific than the best chlamydial cell culture system but offer the advantages of reduced technology and simple transport of clinical specimens. Currently available nonculture tests for C. trachomatis perform adequately as screening tests in populations in which the prevalence of infection is greater than 10%. A negative culture or nonculture test for C. trachomatis does not, however, exclude infection. The predictive value of a positive nonculture test may be unsatisfactory when populations of low infection prevalence are tested. Tests that detect antibody responses to chlamydial infection have limited utility in diagnosis of acute chlamydial infection because of the high prevalence of persistent antibody in healthy adults and the cross-reactivity due to infection by the highly prevalent C. trachomatis and TWAR agents. Assays for changes in antibody titer to the chlamydial genus antigen are used for the diagnosis of respiratory chlamydioses. A single serum sample that is negative for chlamydial antibody excludes the diagnosis of lymphogranuloma venereum.